The most important property of ornamental flowers is flower color. In particular, a yellowish flower color (a color tone ranging from yellow through orange to red) is important. Hitherto, in order to obtain a clear yellowish flower color, breeding has been carried out via crossing. In the cases of roses, older types of garden roses that have been used as ornamental flowers have no yellowish flower color. Thus, a deep yellowish flower color has been imparted to roses via introduction of Rosa foetida that has been found in the West Asia [Hideaki Ooba, Chuko Shinsho, The Birth of Roses (Bara no Tanjo) (1990)]. Also in the case of iris (Iris ensata var. ensata), a yellowish flower color has been imparted thereto via crossing of allied species of Iris pseudacorus [Tsutomu Yabuya, Seibundo Shinkosha Inc., Biohorti, 1, 64-71 (1990)]. However, in the cases of breeding via crossing as described above, the existence of a wild species having yellow color, which can be hybridized, is necessary. In addition, such crossing is very time- and labor-consuming. Actually, in the cases of petunias, asters, gentians, salvias, and the like, which are important horticulture plants, no plants having a clear yellowish color have been obtained through conventional breeding via crossing.
The yellowish flower color is mainly derived from carotenoid or betalain in many cases. In some cases, such color is derived from a flavonoid. In particular, a clear yellowish flower color is often derived from carotenoid.
In the cases of carotenoids (e.g. carotenoids in a broad sense, including xanthophylls having a substituent comprising oxygen [Norio Saito, Seibundo Shinkosha Inc., Biohorti, 1, 49-57 (1990)]), as a gene involved upstream of the carotenoid biosynthetic pathway from farnesyl diphosphate (FPP) through geranylgeranyl pyrophosphate (GGPP) to β-carotene, genes encoding enzyme proteins such as a GGPP synthase derived from Ervinia uredovora, a phytoene synthase, a lycopene synthase, and a β-cyclase have been isolated by Misawa et al. (JP Patent No. 2950888). Thereafter, isolation of carotenoid-metabolic genes from various types of microorganisms or plants has progressed (WO2003/016503). In addition, genes (ketolase genes) involved in the synthesis of ketocarotenoids (e.g., canthaxanthin and astaxanthin) have been obtained by Misawa et al. from microorganisms (JP Patent No. 3375639) and green algae (JP Patent No. 2960967). Also, ketolase genes have been obtained from the yeast Phaffia rhodozyma (U.S. Pat. No. 6,365,386) and plants derived from the Adonis aestivalis (WO99/61625). Further, production of ketocarotenoids (e.g., canthaxanthin and astaxanthin) has been achieved via transformation in microorganisms (Escherichia coli) and yeasts [Miura et al., Appl. Env. Microbiol., 64, 1226-1229 (1998)] with the use of such genes. Also in the cases of plants, it has been attempted to produce carotenoids or increase the amount of carotenoids produced with the use of such genes. There have been reports that carotenoids are stored in rice seeds (rice grains) [Ye et al., Science, 287, 303-305 (2000)], rapeseed seeds [Shewmaker et al, Plant J., 20, 401-412 (1999)], and seeds of Arabidopsis thaliana [Stalberg et al, Plant J., 36, 771-779 (2003)]. However, in these successful examples, carotenoids are stored in seeds. There have been no reports that the amount of carotenoids produced in petals has been successfully increased. Hirshberg et al. expressed a ketolase gene obtained from green algae in tobacco. However, they reported a failure to cause the expression in petals and the expression was found exclusively in a nectary [Mann et al, Nat. Biotechnol., 18, 888-892 (2000)]. Further, cloning of a gene encoding an enzyme protein of a capsanthin-capsorubin synthase causing generation of yellowish pigment of capsicum has been carried out, such synthase being recognized as a key enzyme synthesizing capsanthin or capsorubin known as a ketocarotenoid [Bouvier et al, Plant J., 6, 45-54 (1994)]. However, such gene has not been used for modification of flower color [Davies et al., Acta Hort., 624, 435-447 (2003)].
The aforementioned reports regarding accumulation of carotenoids in rice seeds (rice grains) [Ye et al., Science. 287, 303-305 (2000)], rapeseed seeds [Shewmaker et al., Plant J., 20, 401-412 (1999)], and the like provide examples of instances of increases in the amount of carotenoid produced via introduction of genes of the carotenoid biosynthetic pathway. Also, there are similar reports regarding microorganisms and yeasts [Kajiwara et al., Biochem J., 324, 421-6 (1997); Shimada et al., Appl Environ Microbiol., 64, 2676-80 (1998); Matthews et al., Appl Microbiol Biotechnol., 53, 396-400 (2000); and Kim et al., Biotechnol Bioeng., 72, 408-15 (2001)]. Various types of enzyme genes existing upstream of the pathway for synthesizing carotene (e.g., a phytoene synthase gene, a lycopene synthase gene (phytoene desaturase), an isopentenyl diphosphate isomerase gene, a hydroxymethylglutaryl CoA (HMG-CoA) reductase gene, and a 1-deoxy-D-xylose-5-phosphate synthase gene) have been used in such cases. It is believed that the carotenoid production may be enhanced by different genes depending on hosts. Accordingly, it is shown that rate-limiting processes in a metabolic pathway significantly vary depending on host, thus making it difficult to determine genes that should be enhanced for increasing the amount of carotenoid produced without trial. In particular, there have been no reports clearly suggesting the production of carotenoid in petals. Thus, it is necessary to introduce carotenoid synthase genes upstream of the pathway alone or in combinations of two or more so as to examine whether or not such introduction is effective for causing a yellowish color to be expressed in petals.
In the cases of plants, carotenoid biosynthesis takes place in a plastid, in general. Plastids are known to differentiate into chloroplasts in the cases of cells in which photosynthesis is carried out in a proplastid with the use of the green color of leaves or stems, leucoplasts that store starch and protein in the cases of cells of white tissues constituting roots and the like, or chromoplasts that store carotenoid in the cases of cells of flowers or fruits. It is also known that these are different cell organellas according to morphlogical observation using an electron microscope.
When a protein derived from nuclear DNA is expressed in a chloroplast, a transit peptide is necessary. In order to cause a gene derived from an organism such as a microorganism lacking chloroplast or a gene expressed outside of a chloroplast to be expressed in a chloroplast, the cDNA sequence of a transit peptide, which is a sequence having a function of transferring a gene product to a chloroplast, is ligated to the front of a gene sequence to be expressed. Thus, a gene product is transferred to a chloroplast [Keegstra, Cell, 56, 247-253 (1989)]. In general, the most widely used sequence is a transit peptide derived from a small subunit of ribulose bisphosphate oxygenase/carboxylase protein (RubisCO) of Pisum sativum (garden pea) [Schreier et al., EMBO J., 4, 25-32 (1985) and Misawa et al., Plant J., 4, 833-840 (1993)].
As described above, a transit peptide used for transferring a gene product to a chloroplast has been known. However, there have been no studies of transit peptides involved in carotenoid biosynthesis or protein transport in chromoplasts (particularly in petals). In addition, it is generally believed that transit peptides have no substrate specificity or organ specificity [Jarvis and Soll, Biochemica Biophysica Acta., 1541, 64-79 (2001)]. Thus, there has been an example in which petunias and marigolds were transformed with the use of ketolase genes of microorganisms and green algae obtained by Misawa et al, and it was attempted to cause the expression of such genes in petals (US Patent Publication (Kokai) No. 2004/0003430). In such case, the transit peptide used was a transit peptide derived from a small subunit of RubisCO protein of Pisum sativum, which serves as a signal for the aforementioned transport to a chloroplast. Further, in the above reference, changes in flower color were observed only with the use of a strain obtained by hybridizing transformants into which a phytoene synthase gene and a ketolase gene had been introduced, respectively. Furthermore, based on data from the same reference regarding the transformed plants to which the genes had been separately introduced (tables 12-13), it cannot be said that changes in flower colors in petals of transformed plants were visually observed or that accumulation of pigments developing sufficient colors that are valuable in terms of horticulture took place.
Thus, techniques for causing the expression of genes encoding different enzyme proteins involved in the carotenoid biosynthetic pathway in petals have not been established. Therefore, yellowish flower colors have not been imparted to plants having no or little yellowish flower color. In addition, enhancement of such flower color in plants has not been achieved.
Accordingly, it is an objective of the present invention to provide a means of imparting yellowish flower color to a plant having no or little yellowish flower color or enhancing the yellowish flower color of a plant.